Selectively cleavable linkers have found a number of uses in biochemistry, for example in reversible surface attachment of biomolecules; for pro-drug or targeted drug systems; in synthesis, and particularly in the synthesis of compounds for methods of detection, where the cleavable linker may release, for example, a fluorophore, or a fluorescence quencher moiety.
The detection of biomolecules through specific binding reagents is of great interest for a number of clinical research purposes. Analytes of interest include nucleic acids, such as mRNA, rRNA, genomic DNA, synthetic nucleic acids etc., and polypeptides, particularly antibodies and antigens, hormones, disease markers and the like. In such methods, a sensitive detection system is valuable for detecting minute quantities or concentrations of the analyte of interest. Detection systems of interest include fluorescence-based approaches, for example methods of detecting single nucleotide polymorphisms, genetic discrimination of pathogens, fluorescence activated cell sorting based on genetic variation, genetic identification of specific alleles in vivo, etc. Such systems are also applicable to polypeptide detection, e.g. in the analysis of tissue samples, in the detection of antibodies, pathogens, peptide markers and the like in tissue samples
In particular, cleavable linkers that are triggered with mild, biologically compatible conditions are advantageous for detection of biomolecules in living cells, or samples where disruption of the system is undesirable. For example, detecting nucleic acids or polypeptides directly in living cells holds considerable promise for bioanalytical and clinical assays, as it bypasses time-consuming isolation and amplification steps. Particularly appealing are fluorescence-based approaches that shorten and simplify the protocol by obviating cell fixation and washing steps. Both molecular beacon-based probes and nucleic acid template reactive probes have recently been investigated for this purpose. DNA/RNA templated fluorescence activation, in particular, offers high selectivity, allowing for the discrimination of single nucleotide differences by fluorescence. Recent studies have utilized templated fluorogenic reactions to detect RNAs both in bacterial and mammalian cells.
The possibilities for useful detection and quantitation of specific genes and gene products are nearly endless. Genotyping methods are of interest for prenatal diagnosis; as well as detecting changes in genotype associated with disease, for example during oncogenesis. Genotyping methods also find use in pharmacogenomics, to determine an individual's profile for drug metabolism, including the likelihood of adverse reactions and responsiveness to treatment. Other important areas of research include analysis of mRNA for expression, alternative splicing and SNP variation. In addition to analysis of expression, and of sequence polymorphisms, there is significant interest in simply determining whether a target sequence is present in a sample, for example in the detection and identification of microbial species in clinical and environmental samples.
Evaluation of multiple chemical transformations for templated fluorescence activation has to date revealed several types of reactive probes, however only few are suitable for cellular nucleic acid detection. Quenched autoligation (QUAL) probes rely on an SN2-displacement of a fluorescence quencher to generate a fluorescence turn-on signal. Although QUAL probes allow sensing of highly expressed nucleic acids inside cells and have been used to distinguish between several closely related bacteria, they can be limited by slow ligation and by undesired reactions with endogenous nucleophiles. A second class of templated fluorescence activation probes uses the Staudinger reduction; such probes exhibit rapid kinetics and a high degree of bioorthogonality, beneficial for sensing in cells. However, the reported templated Staudinger schemes have involved the reduction of individually designed profluorophores, thus limiting their versatility and simplicity.
DNA probes comprising cleavable azidoether linkers, linkers that allow for preparation of these probes, and use thereof are described herein.